A Synergistic Pharmaceutical Combination for the Treatment of Squamous Cell
Carcinoma of Head and Neck
Field of Invention:
The present invention relates to a pharmaceutical combination comprising a cyclin
dependent kinase (CDK) inhibitor selected from the compounds of formula I (as described
herein) or a pharmaceutically acceptable salt thereof and one or more antineoplastic agents for
use in the treatment of squamous cell carcinoma of head and neck (SCCHN). The
pharmaceutical combination of the present invention exhibits synergy when used in the treatment
of squamous cell carcinoma of head and neck (SCCHN). Thus, the present invention relates to a
synergistic pharmaceutical combination. The present invention further relates to a
pharmaceutical composition comprising said combination and a method of treating squamous
cell carcinoma of head and neck (SCCHN) in a subject by administrating said pharmaceutical
combination to said subject.
Background of Invention:
Cancer is a group of diseases characterized by the unusual control of cell growth. There
are over 100 different types of cancers, which are classified by the type of cells initially affected
such as bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney
(renal cell) cancer, leukemia, small cell lung cancer, non- small cell lung cancer, pancreatic
cancer, prostate cancer, thyroid cancer, skin cancer, non-hodgkin's lymphoma and melanoma and
head and neck cancer. Squamous cell carcinoma represents more than 90% of all head and
neck cancers. Head and neck squamous cell carcinomas make up the vast majority of head and
neck cancers, and arise from mucosal surfaces throughout the anatomical region. These include
tumors of the nasal cavities, paranasal sinuses, oral cavity, nasopharynx, oropharynx,
hypopharynx, and larynx.
In fact, head and neck cancer (HNC) is the sixth most common cancer worldwide, with
an annual incidence of >640,000 cases worldwide. More than 90% of head and neck cancers are
of squamous histology (HNSCC). Thirty-five percent to 45% of head and neck cancer patients
ultimately die from their disease. In the United States alone, squamous cell carcinoma of the
head and neck comprises about 4% of all malignancies. This corresponds to an
estimated 17 per 100,000 persons with newly diagnosed squamous cell carcinoma of the head
and neck per year (Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008, CA Cancer J. Clin.
2008 Mar-Apr; 58(2):71-96). Squamous cell carcinoma of head and neck (SCCHN) remains a
challenging clinical problem, due to persisting high rate of local and distant failure, as well as
the occurrence of second primaries. Some molecular targeted therapy used in squamous cell
cancers of the head and neck include cetuximab, bevacizumab, erlotinib and reovirus. The best
quality data are available for cetuximab, a recombinant monoclonal antibody, since the 2006
publication of a randomized clinical trial comparing radiation treatment plus cetuximab versus
radiation treatment alone ("Radiotherapy plus cetuximab for squamous-cell carcinoma of the
head and neck". N Engl J Med 2006; 354 (6): 567-78). Another study evaluated the impact of
adding cetuximab to conventional chemotherapy involving use of cisplatin versus cisplatin
alone. This study found no improvement in survival or disease-free survival with the addition
of cetuximab to the conventional chemotherapy (J Clin Oncol. 2005; 23 (34): 8646-54).
However, another study completed in March 2007 found that there was an
improvement in survival. This study is referred to as EXTREME (Erbitux in First-Line
Treatment of Recurrent or Metastatic Head and Neck Cancer) study which is a
European multicenter phase III trial.
Further, it is well established in the art that CDK (Cyclin-dependent kinase) inhibitors
are useful in anti-proliferative therapies for diseases characterized by excessive cell growth
such as cancers and immunological disorders involving unwanted proliferation of leukocytes.
Flavone derivatives useful as CDK inhibitors are described in PCT Patent Publication No.
WO2004-004632 (U.S. Patent 7,271,193) which patent application specifically relates to the
compounds for inhibition of cyclin-dependent kinases, process for their preparation, methods of
inhibiting cyclin-dependent kinases and of inhibiting cell proliferation, use of such compounds
in the treatment of proliferative disorders including cancers. PCT Published application No.
WO2005-053699 (U.S. Patent 7,772,207) relates to a pharmaceutical product comprising a CDK
inhibitor and l-(2-C-cyano-2-dioxy-p-D-arabino-pentofuranosyl)-N4-palmitoyl cytosine or a
metabolite thereof, as a combined preparation for simultaneous, sequential or separate
administration. PCT Published application No. WO2008-122779 (U.S. Patent Appl. Pub. 2010-
0143350) describes combination of CDK inhibitor with a tyrosine kinase inhibitor and use
thereof in the treatment of proliferative disorders. PCT Published application No. WO2008-
139271 (U.S. Patent Appl. Pub. 2010-0305057) relates to pharmaceutical combination
comprising a cytotoxic antineoplastic agent selected from paclitaxel, docetaxel, doxorubicin or
gemcitabine and at least one cyclin dependent kinase (CDK) inhibitor for use in the treatment of
cancer. PCT Published application No. WO2010-128443 describes a combination for the
treatment of cancer wherein the combination comprises radiation and at least one cyclin
dependent kinase (CDK) inhibitor or a pharmaceutically acceptable salt or a solvate thereof.
Although combinations of anticancer agents have been proven to have a significant
advance in various cancer treatment protocols including squamous-cell carcinoma of the head
and neck (SCCHN), there are still several unmet needs and room for improvements in
medications for the treatment of SCCHN, which are difficult to treat, or which have shown
resistance to treatment with the conventional antineoplastic agents. More particularly, the
development of novel combination approach for delivering known anticancer agents having
different mechanism of action would represent an important advance in the art.
Although the protocol involving combination of anticancer agents having different
mechanism of action may work in case of some combinations, it may not work in the same
manner for other combination of anticancer agents and such combination may not always
result in a combination having advantageous therapeutic effects. However, the inventors of the
present invention have found that a pharmaceutical combination of anticancer agents comprising
a cyclin dependant kinase (CDK) inhibitor and one or more antineoplastic agent provides
greater efficacy than when the CDK inhibitors or the antineoplastic agents are used alone for
the treatment of squamous-cell carcinoma of the head and neck (SCCHN).
Summary of the Invention:
According to one aspect of the present invention, there is provided a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of the head and neck
(SCCHN). comprising a cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula (I) or a pharmaceutically acceptable salt thereof and one or more antineoplastic
agents
In another aspect, the present invention provides pharmaceutical compositions for use
the treatment of squamous cell carcinoma of the head and neck (SCCHN), comprising a
combination of a cyclin dependent kinase (CDK) inhibitor selected from the compounds of
formula (I) or a pharmaceutically acceptable salt or solvates thereof and one or more
antineoplastic agents along with at least one pharmaceutically acceptable carrier.
In another aspect, the present invention relates to a method for the treatment of
squamous cell carcinoma of the head and neck (SCCHN) in a subject, comprising administering
to the subject a therapeutically effective amount of a cyclin dependent kinase (CDK) inhibitor
selected from the compounds of formula (I) or pharmaceutically acceptable salts thereof in
combination with a therapeutically effective amount of one or more antineoplastic agents.
According to another aspect, the present invention provides pharmaceutical combination
for use in the treatment of squamous cell carcinoma of the head and neck (SCCHN);
comprising a therapeutically effective amount of a cyclin dependent kinase (CDK) inhibitor
selected from the compounds of formula (I) or a pharmaceutically acceptable salt thereof and a
therapeutically effective amount of one or more antineoplastic agents wherein said
combination exhibits synergistic effect.
In yet another aspect, the present invention relates to a kit comprising a cyclin dependent
kinase (CDK) inhibitor selected from the compounds of formula (I) and one or more
antineoplastic agents; wherein said kit may further include a package insert comprising printed
instructions directing the use of the combined treatment as a method for treating squamous cell
carcinoma of the head and neck.
Other aspects and further scope of applicability of the present invention will become apparent
from the detailed description to follow.
Brief description of the drawings:
Fig. l a is a graphical representation of the percentage inhibition results of dosing of sorafenib
and lapatinib in SCC25 cells.
Fig. lb is a graphical representation of the percentage inhibition results of dosing of compound
A and compound B in SCC25 cells.
Fig. 2a is a graphical representation of the percentage inhibition results of dosing of sorafenib
and lapatinib in Detroit-562 cells
Fig. 2b is a graphical representation of the percentage inhibition results of dosing of
compound A and compound B in Detroit-562 cells.
Fig. 3a is a graphical representation of the percentage inhibition results of dosing of sorafenib
and lapatinib in FADU cells.
Fig. 3b is a graphical representation of the percentage inhibition results of dosing of
compound A and compound B in FADU cells.
Fig. 4a is a graphical representation of the percentage inhibition results of dosing of erlotinib
in Detroit-562 cells.
Fig. 4b is a graphical representation of the percentage inhibition results of dosing of erlotinib
in FADU cells.
Fig. 5a is a graphical representation of the percentage inhibition results of dosing of cisplatin,
5-fluorouracil and docetaxel in Detroit-562 cells.
Fig. 5b is a graphical representation of the percentage inhibition results of dosing of cisplatin,
5-fluorouracil and docetaxel in FADU cells.
Figure 6a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and sorafenib in SCC-25 cells.
Figure 6b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound B and sorafenib in SCC-25 cells.
Figure 7a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and sorafenib in Detroit-562 cells.
Figure 7b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound B and sorafenib in Detroit-562 cells.
Figure 8a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and sorafenib in FADU cells.
Figure 8b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound B and sorafenib in FADU cells.
Figure 9a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and lapatinib in SCC-25 cells.
Figure 9b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound B and lapatinib in SCC-25 cells.
Figure 10a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and lapatinib in Detroit-562 cells.
Figure 10b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound B and lapatinib in Detroit-562 cells.
Figure 11a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and lapatinib in FADU cells.
Figure lib is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound B and lapatinib in FADU cells.
Figure 12a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and erlotinib in Detroit-562 cells.
Figure 12b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A and erlotinib in FADU cells.
Figure 13a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A, cisplatin and 5-FU in Detroit-562 cells.
Figure 13b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A, cisplatin and 5-fluorouracil in FADU cells.
Figure 14a is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A, docetaxel, cisplatin and 5-FU in Detroit-562 cells.
Figure 14b is graphical representation of the percentage cytotoxicity results of single and
combination dosing of compound A, docetaxel, cisplatin and 5-FU in FADU cells.
Figure 15a is graphical representation of activation of Caspase 3 in SCC-25 cells with single
and combination dosing of sorafenib and compound A.
Figure 15b is graphical representation of activation of Caspase 3 in SCC-25 cells with single
and combination dosing of sorafenib and compound B.
Figure 16a is graphical representation of activation of Caspase 3 in SCC-25 cells with single
and combination dosing of lapatinib and compound A.
Figure 16b is graphical representation of activation of Caspase 3 in SCC-25 cells with single
and combination dosing of lapatinib and compound B.
Figure 17a is graphical representation of body weight profile in FaDu xenografts treated with
single and combination dosing of compound A, cisplatin and cetuximab.
Figure 17b is graphical representation of tumor growth inhibition in FaDu xenografts treated
with single and combination dosing of compound A, cisplatin and cetuximab.
Detailed description of the invention:
The present invention encompasses pharmaceutical combinations for use in the
treatment of squamous cell carcinoma of the head and neck (SCCHN), comprising a cyclin
dependent kinase (CDK) inhibitor selected from the compounds of formula (I) (as described
herein) or a pharmaceutically acceptable salt thereof and one or more antineoplastic
agents, wherein said combination exhibits synergistic effect.
According to the present invention there is provided a pharmaceutical composition for
use in the treatment of squamous cell carcinoma of the head and neck (SCCHN) comprising a
therapeutically effective amount of a cyclin dependent kinase (CDK) inhibitor selected from the
compounds of formula (I) or a pharmaceutically acceptable salt thereof and one or more
antineoplastic agents and optionally a pharmaceutically acceptable carrier.
The present invention further provides a method for the treatment of squamous cell
carcinoma of head and neck in a subject, which comprises administering to the said
subject a therapeutically effective amount of a CDK inhibitor selected from the compounds of
formula (I) (as described herein) or a pharmaceutically acceptable salt or solvate thereof;
and a therapeutically effective amount of one or more antineoplastic agents selected
from the group consisting of sorafenib, lapatinib, erlotinib, cisplatin, 5-fluorouracil and
docetaxel or a pharmaceutically acceptable salt thereof; wherein the said CDK inhibitor and the
said antineoplastic agents contained in the combination are administered either simultaneously
or sequentially.
The general terms used hereinbefore and hereinafter preferably have within the context of
this disclosure the following meanings, unless otherwise indicated. Thus, the definitions of the
general terms as used in the context of the present invention are provided herein below:
The singular forms "a," "an," and "the" include plural reference unless the context
clearly dictates otherwise.
The phrase "a cyclin dependent kinase (CDK) inhibitor" or "CDK inhibitor" as used
herein means a compound that exhibits activity against one or more known cyclin
dependent kinases. In the context of the present invention the CDK inhibitor is a pyrrolidine
substituted flavone compound disclosed in PCT Published Application No. WO2004004632,
which application is incorporated herein by reference in its entirety. The CDK inhibitor
according to the present invention is specifically selected from a compound of Formula I as
described herein below or a pharmaceutically acceptable salt or solvate thereof. Further, the
term "CDK inhibitor" as used herein may either refer to the compound of formula I and/or a
pharmaceutically acceptable salt or solvate of the compound of formula I .
The term "antineoplastic agent" is synonymous to "a chemotherapeutic agent" or "an
anticancer agent" and refers to a therapeutic agent, which acts by inhibiting or preventing the
growth of neoplasms. The term "antineoplastic agent" or "anti-cancer agent" in general refers to
the compounds which prevent the cancer cells from multiplying (i.e. anti-proliferative agents).
In general, the antineoplastic agent(s) fall into two classes, anti-proliferative cytotoxic agents
and anti-proliferative cytostatic agents. Cytotoxic agents prevent cancer cells from multiplying
by: (1) interfering with the cell's ability to replicate DNA; and (2) inducing cell death and/or
apoptosis in the cancer cells. The cytostatic agents act via modulating, interfering or inhibiting
the processes of cellular signal transduction which regulate cell proliferation.
The phrase "pharmaceutically acceptable salts" refers to the acid addition salt of
compound of formula I (as described herein) and of an antineoplastic agent, wherein the acid
is selected from an inorganic acid such as hydrochloric acid, hydrobromic acid; or an
organic acid such as benzene sulfonic acid, maleic acid, oxalic acid, fumaric acid, succinic
acid, p-toluenesulfonic acid and maleic acid.
As used herein, the term "combination" or "pharmaceutical combination", means the
combined administration of the anti-cancer agents namely the CDK inhibitor selected from the
compounds represented by formula I and one or more antineoplastic agents which acts by
inhibiting or preventing the growth of neoplasms or the administration of the anti-cancer agents
namely the CDK inhibitor selected from the compounds represented by formula I and the
antineoplastic agents selected from cytostatic or cytotoxic agents; which may be administered
independently at the same time or separately within time intervals that especially allow that the
combination partners to show a synergistic effect.
As used herein, the term "synergistic" or "synergy" means that the effect achieved with
the combinations of anticancer agents encompassed in this invention is greater than the sum
of the effects that result from using anti-cancer agents namely the CDK inhibitor of formula
(I) or a pharmaceutically acceptable salt thereof, antineoplastic agent(s) or a pharmaceutically
acceptable salt thereof, as a monotherapy. Advantageously, such synergy provides greater
efficacy at the same doses, and/or prevents or delays the build-up of multi-drug resistance.
As used herein the term "therapeutically effective amount" in reference to the
treatment of squamous cell carcinoma of head and neck refers to an amount capable of invoking
one or more of the following effects in a subject receiving the combination of the present
invention: (i) inhibition, to some extent, of tumor growth, including, slowing down and
complete growth arrest; (ii) reduction in the number of tumor cells; (iii) reduction in tumor size;
(iv) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into
peripheral organs; (v) inhibition (i.e., reduction, slowing down or complete stopping) of
metastasis; (vi) enhancement of anti-tumor immune response, which may, but does not have to,
result in the regression of the tumor; and/or (vii) relief, to some extent, of one or more
symptoms associated with squamous cell carcinoma of head and neck (SCCHN).
The term "subject" as used herein, refers to an animal, preferably a mammal, most
preferably a human, who has been the object of treatment, observation or experiment. The term
subject may be interchangeably used with the term "patient" in the context of the present
invention.
As used herein, the term "simultaneously" means that two or more therapeutic agents
(anticancer agents) are administered concurrently, "sequentially" means that two or more
therapeutic agents are available to act therapeutically within the same time-frame and
"separately" means that the gap between administering one agent and the other is significant i.e.
the first administered agent may no longer be present in the bloodstream in a therapeutically
effective amount when the second agent is administered.
The term "caspase3 activity" as used herein refers to increase in apoptosis in cancer
cells.
The term "apoptosis" refers to a type of cell death in which a series of molecular steps in
a cell leads to its death. This is the body's normal way of getting rid of unneeded or abnormal
cells. The process of apoptosis may be blocked in cancer cells. Also called programmed cell
death. (Dictionary of cancer terms, National Cancer Institute). The term "increasing apoptosis"
is defined as an increase in the rate of programmed cell death, i.e. more cells are induced into
the death process as compared to exposure (contact) with either the antineoplastic agent alone or
the CDK inhibitor alone.
The phrase "pharmaceutically acceptable carrier" refers to one or more disintegrating
agents, binders, excipients, lubricants and the like which are well known to those skilled in
the art.
In the present invention there is provided a pharmaceutical combination of anti-cancer
agents for use in the treatment of squamous cell carcinoma of head and neck (SCCHN). The
present inventors have conducted an extensive research for the development of the
pharmaceutical combination of anti-cancer agents and arrived at the present synergistic
pharmaceutical combination. It has been found that pharmaceutical combination comprising a
cyclin dependent kinase (CDK) inhibitor selected from the compounds of formula I or a
pharmaceutically acceptable salt thereof and one or more antineoplastic agent exhibits
synergistic effect when used in the treatment of squamous cell carcinoma of the head and neck
(SCCHN).
The CDK inhibitor is a pyrrolidine substituted flavone compound that inhibits cyclin
dependent kinases. The CDK inhibitor used in the pharmaceutical combination of the present
invention is selected from the compounds of formula I or pharmaceutically acceptable salts or
solvates thereof as described herein below. The compounds of formula I are promising CDK
inhibitors, which can inhibit proliferation of many cancer cells. As indicated herein above the
CDK inhibitors of formula (I) may be used in the form of their pharmaceutically acceptable
salts. The salts encompassed within the term "pharmaceutically acceptable salts" refer to non
toxic salts of the compounds of this invention. Representative salts include, but are not limited
to acetate, benzoate, benzenesulfonate, bicarbonate, chloride, citrate,
hydrochloride, mesylate, methylsulfonate, tartrate, tosylate and trifluoroacetate. Preferred salts
of compounds of formula (I) include hydrochloride salt, methanesulfonic acid and
trifluoroacetic acid salt.
In one embodiment, the CDK inhibitor used in the pharmaceutical combination of the
present invention is selected from the compounds represented by the following formula I,
Formula I
wherein, Ar is a phenyl group, which is unsubstituted or substituted by 1, 2, or 3 identical or
different substituents selected from : halogen, nitro, cyano, C1-C4-alkyl, trifhioromethyl,
hydroxyl or C1-C4-alkoxy; or a pharmaceutically acceptable salt or solvate thereof.
As indicated herein above the salts of CDK inhibitor refers to non-toxic salts of the
compounds of formula (I) of this invention. Representative salts include, but are not limited to
acetate, benzoate, benzenesulfonate, bicarbonate, chloride, citrate, hydrochloride, mesylate,
methylsulfonate, tartrate, tosylate and trifluoroacetate. Preferred salts of compounds of
formula (I) include hydrochloride salt, methanesulfonic acid and trifluoroacetic acid salt.
In an embodiment of the invention, the CDK inhibitor is the (+)-trans isomer of the
compound of formula I, as indicated in Formula IA below,
F la
wherein Ar is a phenyl group, which is unsubstituted or substituted by 1, 2, or 3 identical or
different substituents selected from :halogen, nitro, cyano, Ci-C 4-alkyl, trifluoromethyl,
hydroxyl or Ci-C4-alkoxy; or a pharmaceutically acceptable salt or solvate thereof.
In another embodiment of the present invention, the CDK inhibitor used in the
pharmaceutical combination of the present invention is a compound of formula I wherein the
phenyl group is substituted by 1, 2 or 3 identical or different substituents selected from:
chlorine, bromine, fluorine or iodine, Ci-C 4-alkyl or trifluoromethyl; or a pharmaceutically
acceptable salt or solvate thereof.
In another embodiment of the present invention, the CDK inhibitor used in the
pharmaceutical combination of the present invention is a compound of formula I wherein the
phenyl group is substituted by chlorine; or a pharmaceutically acceptable salt or solvate thereof.
In another embodiment of the present invention, the CDK inhibitor used in the
pharmaceutical combination of present invention is a compound of formula I wherein the
phenyl group is substituted by two different substituents selected from chlorine and
trifluoromethyl; or a pharmaceutically acceptable salt or solvate thereof.
It will be appreciated by those skilled in the art that the CDK inhibitors represented by
the compounds of formula (I) contain at least two chiral centers and hence, exist in the form of
two different optical isomers (i.e. (+) or (-) enantiomers). All such enantiomers and mixtures
thereof including racemic mixtures are included within the scope of the invention. The
enantiomers of the compound of formula I can be obtained by methods disclosed in PCT
Application Publication No. WO2004004632 incorporated herein by reference or the
enantiomers of the compound of formula I can also be obtained by methods well known in
the art, such as chiral HPLC and enzymatic resolution.
Alternatively, the enantiomers of the compounds of formula I can be synthesized by
using optically active starting materials. The manufacture of the compounds of formula I,
which may be in the form of pharmaceutically acceptable salts and solvates, and the manufacture
of oral and/or parenteral pharmaceutical composition containing the above compounds are
generally disclosed in PCT Application Publication No. WO2004004632. This patent
application, which is incorporated herein by reference, discloses that the CDK inhibitors
represented by formula I exhibit significant anticancer efficacy. As indicated herein above
the CDK inhibitors of formula I may be used in the form of their salts. Preferred salts of
compounds of formula I include hydrochloride, methanesulfonic acid and trifluoroacetic acid
salt.
According to another embodiment of the present invention, the CDK inhibitor used in
the pharmaceutical combination of the present invention is selected from (+)-trans-2- (2-
Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-l-methyl-pyrrolidin-3-yl)- chromen-4-one
hydrochloride (referred to herein as compound A) or (+)-trans-3-[2[(2- Chloro-4-
trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl- pyrrolidin-3-yl)-chromen-
4-one hydrochloride (referred to herein as compound B).
In an embodiment of the present invention, the CDK inhibitor used in the
pharmaceutical combination is (+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2- hydroxymethyl-
1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (compound A).
In further embodiment of the present invention, the CDK inhibitor used in the
pharmaceutical combination is (+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-
dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(compound B).
The antineoplastic agents are the compounds that prevent cancer cells from
multiplying (i.e. anti-proliferative agents). In the present invention anti-neoplastic agent
included in the pharmaceutical combination may be selected from either cytostatic or cytotoxic
agents.
According to an embodiment of the invention, an anti-neoplastic agent used in the
pharmaceutical combination of the present invention is a cytostatic agent.
According to another embodiment of the invention, an anti-neoplastic agent used in the
pharmaceutical combination of the present invention is a cytotoxic agent.
According to an embodiment of the invention, when the anti-neoplastic agent used in
the pharmaceutical combination of the present invention is a cytostatic agent, it is selected from
small molecules such as sorafenib, lapatinib or erlotinib or a chimeric monoclonal antibody such
as cetuximab.
According to another embodiment of the invention, when the anti-neoplastic agent
used in the pharmaceutical combination of the present invention is a cytotoxic agent, it is
selected from cisplatin, 5-fluorouracil and/or docetaxel or pharmaceutically acceptable salts
thereof.
According to another embodiment of the invention, the pharmaceutical combination
comprising a CDK inhibitor selected from the compounds of formula (I) or a pharmaceutically
acceptable salt thereof, and one or more antineoplastic agents, may further include use of
radiation therapy for the treatment of squamous cell carcinoma of the head and neck (SCCHN).
The specified anti-neoplastic agents used in the present invention are commercially
readily available.
Sorafenib is a kinase inhibitor that decreases tumor cells proliferation in vitro.
Sorafenib was shown to inhibit multiple intracellular (CRAF, BRAF and mutant BRAF)
and cell surface kinases (KIT, FLT-3, RET, VEGFR-1 to 3 and PDGFR- . Several of these
kinases are thought to be involved in tumor cell signaling, angiogenesis and apoptosis.
Sorafenib inhibited tumor growth and angiogenesis of human hepatocellular carcinoma and
renal cell carcinoma and several other human tumor xenografts in immunocompromised mice.
It is chemically named as 4-(4-{3-[4-
chloro-3-(trifluoromethyl)phenyl]ureido }phenoxy)N -methylpyridine-2-carboxamide-
4-methylbenzenesulfonate. Sorafenib is commercially available and is marketed as Nexavar
® by Bayer in the United States for the treatment of patients with advanced renal cell carcinoma
(RCC) and those with unresectable hepatocellular carcinoma (HCC). It is also approved by the
European Medicines Agency for the treatment of patients with HCC and patients with
advanced RCC with whom prior IFN-CC or interleukin-2-based therapy had failed or considered
to be unsuitable for such therapy ("Preclinical overview of sorafenib, a multikinase inhibitor
that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling". Molecular
Cancer Therapeutics2008; 7 (10): 3129-40).
Lapatinib, is a 4-anilinoquinazoline kinase inhibitor of the intracellular tyrosine kinase
domains of both Epidermal Growth Factor Receptor (EGFR [ErbBl]) and of human Epidermal
Receptor Type 2 (HER2[ErbB2]) receptors. Lapatinib inhibits ErbB-driven tumor cell growth in
vitro and in various animal models. It is present as the monohydrate of the ditosylate salt, with
chemical name N-(3-chloro-4-{[(3- fluorophenyl) methyl]oxy}phenyl)-6-[5-({[2-
(methylsulfonyl) ethyl] amino}methyl)- 2-furanyl]-4-quinazolinaminebis(4-
methylbenzenesulfonate)monohydrate. Lapatinib ditosylate monohydrate is a dual tyrosine
inhibitor which interrupts the HER2 growth receptor and is used in combination therapy for
HER2-positive breast cancer ("Lapatinib in the treatment of breast cancer" Expert Review of
Anticancer Therapy (Future Drugs) 7 (9): 1183-92). It is marketed under the brand name
TYKERB® in the United States by GlaxoSmithKline and is available commercially.
Lapatinib inhibits the tyrosine kinase activity associated with two oncogenes, EGFR (epidermal
growth factor receptor) and HER2/neu (Human EGFR type 2) ("A unique structure for epidermal
growth factor receptor bound to GW572016 (Lapatinib): relationships among protein
conformation, inhibitor off-rate, and receptor activity in tumor cells" Cancer Res. 2004 Sep 15;
64(18): 6652-9). Lapatinib inhibits receptor signal processes by binding to the ATP-binding
pocket of the EGFR/HER2 protein kinase domain, preventing self-phosphorylation and
subsequent activation of the signal mechanism ("Lapatinib: a novel dual tyrosine kinase
inhibitor with activity in solid tumors". Annals of Pharmacotherapy: 40 (2); 261-269).
Erlotinib is an EGFR inhibitor. The drug follows gefitinib (Iressa®), which was the
first drug of this type. Gefitinib and erlotinib are commercially available epidermal growth
factor receptor tyrosine kinase inhibitors (EGFR-TKIs) that are widely used for the treatment of
non-small-cell lung cancer (NSCLC). Erlotinib specifically targets the epidermal growth factor
receptor (EGFR) tyrosine kinase, which is highly expressed and occasionally mutated in various
forms of cancer. It binds in a reversible fashion to the adenosine triphosphate (ATP) binding site
of the receptor (J Clin Oncol, 2007;25:1960-1966).
Cisplatin is a platinum compound which acts as a cytotoxic anticancer agent. This
platinum-based chemotherapy drug, which kills the cancer cells by damaging DNA and
inducing apoptosis. Cisplatin is commercially available for the treatment of various types of
cancers, including sarcomas, some carcinomas (e.g. small cell lung cancer, and ovarian
cancer), lymphomas, and germ cell tumors. Cisplatin is a non cell cycle specific cytotoxic agent
which is effective against cells that are actively dividing as well as those that are merely resting
before entering the cell cycle and reacts in vivo, binding to and causing cross linking of DNA
which ultimately triggers apoptosis (programmed cell death).
Fluorouracil (5-FU) is an antimetabolite and a cytotoxic anti-cancer agent. 5-FU inhibits
DNA synthesis and cell death and penetrates cerebrospinal fluid well. 5-FU is commercially
available as an antimetabolite that interferes with RNA and DNA synthesis. 5-FU is
therapeutically useful for certain types of carcinoma, such as carcinoma of the colon, rectum,
breast, stomach and pancreas.
Docetaxel is an antineoplastic agent belonging to the taxoid family that acts by
disrupting the microtubular network in cells that is essential for mitotic and interphase cellular
functions. Docetaxel binds to free tubulin and promotes the assembly of tubulin into
stable microtubules while simultaneously inhibiting their disassembly. This leads to the
production of microtubule bundles without normal function and to the stabilization of
microtubules, which results in the inhibition of mitosis in cells. Docetaxel's binding to
microtubules does not alter the number of protofilaments in the bound microtubules, a feature
which differs from most spindle poisons currently in clinical use. Docetaxel is marketed
worldwide under the name Taxotere® by Sanofi- and available commercially.
Cetuximab is a recombinant, chimeric monoclonal antibody directed against the
epidermal growth factor (EGFR) with antineoplastic activity. Cetuximab binds to the
extracellular domain of the EGFR, thereby preventing the activation and subsequent
dimerization of the receptor; the decrease in receptor activation and dimerization may result in
an inhibition in signal transduction and anti-proliferative effects. This agent may inhibit EGFRdependent
primary tumor growth and metastasis. Cetuximab is commercially available as
Erbitux® for treatment of metastatic colorectal cancer and head and neck cancer.
According to another embodiment, the present invention relates to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck (SCCHN)
wherein the combination comprises a cyclin dependent kinase (CDK) inhibitor selected
from the compounds of formula I or a pharmaceutically acceptable salt or a solvate thereof and
one or more of antineoplastic agents selected from sorafenib, lapatinib, erlotinib, cisplatin, 5-
fluorouracil or docetaxel or a pharmaceutically acceptable salt thereof or the monoclonal
antibody cetuximab.
In another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck (SCCHN)
wherein the combination comprises a cyclin dependent kinase (CDK) inhibitor selected
from the compounds of formula I or a pharmaceutically acceptable salt or a solvate thereof and
sorafenib.
Another embodiment of the present invention provides a pharmaceutical combination for
use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises a cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula I or a pharmaceutically acceptable salt or a solvate thereof and lapatinib.
In another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises a cyclin dependent kinase (CDK) inhibitor selected from the
compounds of formula I or a pharmaceutically acceptable salt or a solvate thereof and erlotinib.
Another embodiment of the present invention provides a pharmaceutical combination for
use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises a cyclin dependent kinase (CDK) inhibitor selected from the compounds
of formula I or a pharmaceutically acceptable salt or a solvate thereof; cisplatin and 5-
fluorouracil or a pharmaceutically acceptable salt thereof.
In another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein
the combination comprises a cyclin dependent kinase (CDK) inhibitor selected from the
compounds of formula I or a pharmaceutically acceptable salt or a solvate thereof;
docetaxel, cisplatin and 5-fluorouracil or a pharmaceutically acceptable salt thereof.
Further embodiment of the present invention provides a pharmaceutical combination
for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises a CDK inhibitor selected from compound A or compound B and one
or more anti-neoplastic agents selected from sorafenib, lapatinib, erlotinib, cisplatin, 5-
fluorouracil or docetaxel or a pharmaceutically acceptable salt thereof.
In another embodiment, the present invention provides a pharmaceutical combination for
use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises compound A and sorafenib or a pharmaceutically acceptable salt
thereof.
In another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises compound A and lapatinib or a pharmaceutically acceptable salt
thereof.
In another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises compound A and erlotinib or a pharmaceutically acceptable salt
thereof.
In another embodiment, the present invention is directed to pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises compound A, cisplatin and 5-fluorouracil or a pharmaceutically
acceptable salt thereof.
Further embodiment of the present invention is directed to pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises compound A, docetaxel, cisplatin and 5-fluorouracil or a
pharmaceutically acceptable salt thereof.
In another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises compound B and sorafenib or a pharmaceutically acceptable salt
thereof.
In another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck wherein the
combination comprises the compound B and lapatinib or a pharmaceutically acceptable salt
thereof.
According to another embodiment of the present invention, the pharmaceutical
combination comprising the CDK inhibitor selected from the compounds of formula I and an
antineoplastic agent selected from sorafenib, lapatinib or erlotinib or the pharmaceutical
combination comprising the CDK inhibitor selected from the compounds of formula I, and an
antineoplastic agent selected from cisplatin and 5-fluorouracil or the pharmaceutical
combination comprising the CDK inhibitor selected from the compounds of formula I, and an
antineoplastic agent selected from cisplatin, 5-fluorouracil and docetaxel, is not exclusively
limited to those combinations which are obtained by physical association of said ingredients, but
also encompass those which permit a separate administration, which can be simultaneous,
sequential or spaced out over a period of time so as to obtain maximum efficacy of the
combination. Thus, the pharmaceutical combination may be administered simultaneously or
sequentially for an effective treatment of squamous cell carcinoma of head and neck.
According to another embodiment, the present invention is directed to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma comprising radiation, a CDK
inhibitor selected from the compounds of formula I and one or more antineoplastic agents
selected from sorafenib, lapatinib, erlotinib, cisplatin, 5-fluorouracil or docetaxel or a
pharmaceutically acceptable salt thereof.
According to another embodiment, the present invention is directed to a
pharmaceutical combination for use in the treatment of squamous cell carcinoma comprising
radiation, a CDK inhibitor selected from compound A or compound B and one or more
antineoplastic agents selected from sorafenib, lapatinib, erlotinib, cisplatin, 5- fluorouracil or
docetaxel or a pharmaceutically acceptable salt thereof.
According to another embodiment, the present invention relates to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck (SCCHN)
wherein the combination comprises a cyclin dependent kinase (CDK) inhibitor selected
from compound A or compound B, cisplatin and or a pharmaceutically acceptable salt thereof
and the monoclonal antibody, cetuximab.
According to another embodiment, the present invention relates to a pharmaceutical
combination for use in the treatment of squamous cell carcinoma of head and neck (SCCHN)
wherein the combination comprises radiation, a cyclin dependent kinase (CDK) inhibitor
selected from compound A or compound B or a pharmaceutically acceptable salt thereof and the
monoclonal antibody, cetuximab.
In further embodiment, the present invention provides a pharmaceutical composition
which comprises a therapeutically effective amount of a CDK inhibitor selected from the
compounds of formula I (as described herein) or a pharmaceutically acceptable salt or solvate
thereof in combination with a therapeutically effective amount of one or more antineoplastic
agents selected from the group consisting of sorafenib, lapatinib, erlotinib, docetaxel,
cisplatin and 5-fluorouracil or a pharmaceutically acceptable salt thereof; in association with a
pharmaceutically acceptable carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of a CDK inhibitor selected from the
compounds of formula I or a pharmaceutically acceptable salt or solvate thereof and a
therapeutically effective amount of sorafenib in association with a pharmaceutically acceptable
carrier.
In another further embodiment, the present invention relates to a pharmaceutical
composition which comprises a therapeutically effective amount of a CDK inhibitor selected
from the compounds of formula I or a pharmaceutically acceptable salt or solvate thereof
and a therapeutically effective amount of lapatinib in association with a pharmaceutically
acceptable carrier.
In another further embodiment, the present invention relates to a pharmaceutical
composition which comprises a therapeutically effective amount of a CDK inhibitor selected
from the compounds of formula I or a pharmaceutically acceptable salt or solvate thereof
and a therapeutically effective amount of erlotinib in association with a pharmaceutically
acceptable carrier.
In further embodiment, the present invention provides a pharmaceutical composition
which comprises a therapeutically effective amount of a CDK inhibitor selected from the
compounds of formula I or a pharmaceutically acceptable salt or solvate thereof, a
therapeutically effective amount of cisplatin and a therapeutically effective amount of 5-
fluorouracil or a pharmaceutically acceptable salt thereof; in association with a
pharmaceutically acceptable carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of a CDK inhibitor selected from the
compound A or compound B and therapeutically effective amount of one or more antineoplastic
agents or a pharmaceutically acceptable salt thereof; in association with a pharmaceutically
acceptable carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of a CDK inhibitor selected from compound
A or compound B and a therapeutically effective amount of one or more antineoplastic agents
selected from the group consisting of sorafenib, lapatinib, erlotinib, cisplatin and 5-
fluorouracil or pharmaceutically acceptable salt thereof; in association with a pharmaceutically
acceptable carrier.
In another further embodiment, the present invention relates to a pharmaceutical
composition which comprises a therapeutically effective amount of compound A and a
therapeutically effective amount of sorafenibin association with a pharmaceutically acceptable
carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of compound A and a therapeutically
effective amount of lapatinib in association with a pharmaceutically acceptable carrier.
In further embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of compound B and a
therapeutically effective amount of sorafenib in association with a pharmaceutically acceptable
carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of compound B and a therapeutically
effective amount of lapatinib in association with a pharmaceutically acceptable carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of compound A and a therapeutically
effective amount of erlotinib in association with a pharmaceutically acceptable carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of each of the compound A, cisplatin and
5-fluorouracil or a pharmaceutically acceptable salt thereof; in association with a
pharmaceutically acceptable carrier.
In another embodiment, the present invention relates to a pharmaceutical composition
which comprises a therapeutically effective amount of each of the compound A, docetaxel,
cisplatin and 5-fluorouracil or a pharmaceutically acceptable salt thereof; in association with a
pharmaceutically acceptable carrier.
In further embodiment, the present invention is directed to a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises administering to said
subject a therapeutically effective amount of a CDK inhibitor selected from the compounds
of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a therapeutically
effective amount of an anti-neoplastic agent selected from sorafenib, lapatinib or erlotinib;
wherein said CDK inhibitor and said anti-neoplastic agent or pharmaceutically acceptable salt
thereof is administered simultaneously or sequentially.
In another embodiment, the present invention is directed to a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises administering to said
subject a therapeutically effective amount of a CDK inhibitor selected from the compounds of
formula (I) or a pharmaceutically acceptable salt or solvate thereof; a therapeutically effective
amount of each of cisplatin and 5- fluorouracil or a pharmaceutically acceptable salt thereof;
wherein said CDK inhibitor, cisplatin and 5-fluorouracil are administered simultaneously or
sequentially.
In further embodiment, the present invention is directed to a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises administering to said
subject a therapeutically effective amount of a CDK inhibitor selected from the compound A
or compound B and a therapeutically effective amount of antineoplastic agent selected from
sorafenib, lapatinib or erlotinib; wherein said compound A or compound B and antineoplastic
agent is administered simultaneously or sequentially.
In another embodiment, the present invention is directed to a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises administering to said
subject a therapeutically effective amount of a CDK inhibitor selected from the compound A
or compound B and a therapeutically effective amount of sorafenib; wherein said compound A
or compound B and sorafenib is administered simultaneously or sequentially.
In another embodiment, the present invention is directed to a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises administering to said
subject a therapeutically effective amount of a CDK inhibitor selected from the compound A
or compound B and a therapeutically effective amount of lapatinib or a pharmaceutically
acceptable salt thereof; wherein said compound A or compound B and lapatinib is administered
simultaneously or sequentially.
Another embodiment of the present invention provides a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises
administering to said subject a therapeutically effective amount of compound A or a
pharmaceutically acceptable salt or solvate thereof and a therapeutically effective amount of
erlotinib; wherein said compound A and erlotinib is administered simultaneously or
sequentially.
In another embodiment, the present invention is directed to a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises administering to said
subject a therapeutically effective amount of compound A or a pharmaceutically acceptable salt
or solvate thereof; a therapeutically effective amount of cisplatin and 5-fluorouracil or a
pharmaceutically acceptable salt thereof; wherein said compound A, cisplatin and 5-fluorouracil
are administered simultaneously or sequentially.
Another embodiment of the present invention is directed to a method for the treatment of
squamous cell carcinoma of head and neck in a subject, which comprises administering to said
subject a therapeutically effective amount of CDK inhibitor selected from the compounds of
formula (I) or a pharmaceutically acceptable salt or solvate thereof and a therapeutically
effective amount of an antineoplastic agent or a pharmaceutically acceptable salt thereof;
wherein said CDK inhibitor and said anti-neoplastic agent or their pharmaceutically
acceptable salts are administered sequentially.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound A or compound B; a
therapeutically effective amount of an antineoplastic agent selected from sorafenib, lapatinib
or erlotinib; wherein said compound A or compound B and antineoplastic agent selected from
sorafenib, lapatinib or erlotinib is administered sequentially such that compound A or
compound B is administered before or after the administration of sorafenib or lapatinib or
erlotinib.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound A and a therapeutically effective
amount of sorafenib; wherein said compound A and sorafenib is administered sequentially such
that compound A is administered before or after the administration of sorafenib.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound B and a therapeutically effective
amount of sorafenib; wherein said compound B and sorafenib is administered sequentially such
that compound B is administered before or after the administration of sorafenib.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound A and a therapeutically effective
amount of lapatinib; wherein said compound A and lapatinib is administered sequentially such
that compound A is administered before or after the administration of lapatinib.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound B and a therapeutically effective
amount of lapatinib; wherein said compound B and lapatinib is administered sequentially such
that compound B is administered before or after the administration of lapatinib.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound A; a therapeutically effective
amount of erlotinib; wherein said compound A and erlotinib is administered sequentially such
that compound A is administered before or after the administration of erlotinib.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound A; a therapeutically effective
amount of each of cisplatin; and 5-fluorouracil or a pharmaceutically acceptable salt
thereof; wherein said compound A, cisplatin and 5- fluorouracil or a pharmaceutically
acceptable salt thereof are administered sequentially such that compound A is administered
before or after the administration of cisplatin and/or 5-fluorouracil.
Accordingly to another embodiment, the present invention relates to a method for the
treatment of squamous cell carcinoma of head and neck in a subject comprising administering to
said subject a therapeutically effective amount of compound A; a therapeutically effective
amount of each of docetaxel; cisplatin and 5-fluorouracil or a pharmaceutically acceptable salt
thereof; wherein said compound A, docetaxel, cisplatin and 5-fluorouracil or a pharmaceutically
acceptable salt thereof are administered sequentially such that compound A is administered
before or after the administration of docetaxel, and/or cisplatin and/or 5-fluorouracil.
In another embodiment the present invention provides use of combination of a CDK
inhibitor selected from the compound of formula I or pharmaceutically acceptable salt or solvate
thereof and one or more antineoplastic agents or a pharmaceutically acceptable salt thereof for
the manufacture of a medicament for the treatment or prevention of squamous cell carcinoma of
the head and neck (SCCHN).
Another embodiment of the present invention provides use of pharmaceutical
composition comprising a therapeutically effective amount of CDK inhibitor selected from the
compounds of formula (I) or a pharmaceutically acceptable salt thereof and an antineoplastic
agent for the manufacture of a medicament for the treatment of squamous cell carcinoma of the
head and neck (SCCHN).
According to the present invention the administration of the double combination of
CDK inhibitor selected from the compound of formula I and an antineoplastic agent or a
pharmaceutically acceptable salt thereof selected from sorafenib, lapatinib or erlotinib
may produce effects, such as the anti-cancer effects , greater than those achieved with any of
the CDK inhibitor or sorafenib or lapatinib or erlotinib when used alone.
It is further provided by the present invention that the administration of a triple
combination of the CDK inhibitor selected from the compound of formula I as described herein,
cisplatin and 5-fluorouracil may produce effects, such as anti-cancer effects, greater than those
achieved with any of the CDK inhibitor or cisplatin or 5- fluorouracil used alone, greater than
those achieved with the combination of the CDK inhibitor, cisplatin and 5-fluorouracil.
The administration route of the pharmaceutical composition of the present invention is
not particularly limited. In one embodiment, the active ingredients (the anticancer agents
contained in the combination) comprised in the composition may have to be administered by
different routes either orally or parenterally depending on the dosage form. The dosage form
suitable for oral administration may be a tablet or capsule, forms of parenteral administration
include intravenous injection, intravenous infusion, subcutaneous injection, transdermal
injection, intraperitoneal injection and so on. For rectal administration, for example as a
suppository or the route of administration may be by direct injection into the tumour or by
regional delivery or by local delivery. In the case of tablets for oral use, carriers which are
commonly used include lactose, corn starch, magnesium carbonate, talc, and sugar, and
lubricating agents such as magnesium stearate are commonly added. For oral administration in
capsule form, useful carriers include lactose, corn starch, magnesium carbonate, talc and sugar.
For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the
active ingredient are usually employed, and the pH of the solutions should be suitably adjusted
and buffered.
In practice of the present invention, CDK inhibitors selected from the compounds of
Formula I may be administered either orally or parenterally to generate and maintain good
blood levels thereof, while one or more antineoplastic agents may be administered
orally or parenterally, by intravenous, subcutaneous or intramuscular route or any other
suitable route of administration.
In one embodiment, the therapeutic agents (the CDK inhibitors and the antineoplastic
agents) contained in the combination of the invention are formulated in accordance with
routine procedures as a pharmaceutical composition.
In practice, oral preparations for oral administration may be produced by adding to the
active ingredients fillers, and if necessary, binders, disintegrants, lubricants, coloring agents,
flavoring agents, etc. and formulating the resultant mixture according to conventional
procedures into tablets, coated tablets, granules, subtle granules, powders, capsules or the
like. Examples of the filler include but not limited to lactose, corn starch, white sugar, glucose,
sorbitol, crystalline cellulose, silicon dioxide, etc. Examples of the binder include but not
limited to polyvinyl alcohol, ethylcellulose, methylcellulose, gum arabic, hydroxypropyl
cellulose, hydroxypropyl methylcellulose, etc. Examples of the lubricant include but not
limited to magnesium stearate, talc, silica, etc. The coloring agent may be any coloring
agent which is approved to be added to pharmaceutical preparations. Examples of the flavoring
agent include but not limited to cocoa powder, menthol, aromatic powder, peppermint oil,
camphol, cinnamon powder, etc. Resultant tablets and granules may be appropriately coated
with, for example, sugar or gelatin according to necessity. When the pharmaceutical
composition of the present invention is administered transdermally in the form of patch, it
is preferable to select the so-called free-form that does not form a salt. Injection preparations
may be produced as intravenous infusion preparations or intravenous, subcutaneous or
intramuscular injection preparations according to conventional procedures. Examples of the
suspending agent include but not limited to methylcellulose, polysolvate 80, hydroxyethyl
cellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene
sorbitan monolaurate, etc. Examples of the dissolution aid include but not limited to
polyoxyethylene hydrogenated castor oil, polysolvate 80, nicotinamide, polyoxyethylene
sorbitan monolaurate, macrogol, fatty acid ethyl ester from castor oil, etc. Examples of the
stabilizer include but not limited to sodium sulfite, sodium metasulfite, etc. Examples of the
preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid,
phenol, cresol, chlorocresol etc.
Although the effective doses of therapeutic agents (the CDK inhibitors and the
antineoplastic or anticancer agents) for administration vary depending on the severity of
symptom, the age, sex, body weight and sensitivity difference of the patient, the mode, time,
interval and duration of administration, the nature, formulation and type of the preparation, the
type of the active ingredient, etc. In certain embodiments, the therapeutic agents are
administered in a time frame where both agents are still active. One skilled in the art would
be able to determine such a time frame by determining the half life of the administered
therapeutic agents. As indicated herein before, the active ingredients contained in the
pharmaceutical composition can be administered simultaneously or sequentially. Those skilled
in the art will recognize that several variations are possible within the scope and spirit of this
invention.
For effective administration, the therapeutic agents of the pharmaceutical combination of
the present invention are provided in a particular dose range, for example the CDK inhibitor
selected from compound of formula I such as the compound A may be provided in a general
dose range of 75 mg/m2/day to 200 mg/m2/day; another CDK inhibitor selected from
compound of formula I such as the Compound B may be provided in a general dose range of
50 mg to 350 mg orally. Further, among the antineoplastic agents, cisplatin may be
provided in a dose range of 40 mg/m2/day to 200 mg/m2/day, 5-fluourouracil may be provided
in dose range of 40 mg/m2/day to 200 mg/m2/day , docetaxel may be provided in a general dose
range of 20 mg/m2/day to 75 mg/m2/day, sorafenib may be provided in at least an amount from
about 200 mg to 400mg (2x200 mg tablet) PO bid (orally twice a day), lapatinib may be
provided in a dose ranging from 500 to 1500 mg/d and erlotinib may be provided in a dose
range of about 150 mg/day to 300 mg/day.
In a further embodiment, the present invention provides a kit comprising a
therapeutically effective amount of a CDK inhibitor selected from the compound of formula I
or a pharmaceutically acceptable salt thereof in combination with one or more antineoplastic
agents selected from sorafenib, lapatinib, erlotinib, cisplatin and 5- fluorouracil or a
pharmaceutically acceptable salt thereof.
The combinations provided by this invention have been evaluated in certain assay
systems, the experimental details are as provided herein below.
The synergistic efficacy of the combination of present invention is demonstrated by
conducting the in vitro study involving use of a combination for example a CDK inhibitor of
formula I as described herein as compound A or compound B and one or more antineoplastic
agents selected from sorafenib, lapatinib, erlotinib, cisplatin, 5- fluorouracil or docetaxel. It
is clearly indicated that the antineoplastic agents when used in combination with CDK
inhibitors in the treatment of squamous cell carcinoma of head and neck the apoptosis in
proliferative cells increases than when the cells are treated with the CDK inhibitor of formula I
alone or antineoplastic agent alone. For instance, it is clearly established from the data described
herein that the CDK inhibitor of formula I, compound described herein as the compound A or
compound B in combination with one or more antineoplastic agents selected from sorafenib,
lapatinib, erlotinib, cisplatin, 5-fluorouracil or docetaxel, are synergistically effective in the
treatment of squamous cell carcinoma of the head and neck. The synergism exhibited by the
pharmaceutical combination of the present invention is also demonstrated through in vivo study
data as indicated herein.
It is understood that modifications that do not substantially affect the activity of the various
embodiments of this invention are included within the invention disclosed herein. Accordingly,
the following examples are intended to illustrate but not to limit the present invention.
Example 1:
A) General procedure for the preparation of the CDK inhibitors (the compounds of
Formula I):
The compounds of formula I may be prepared according to the methods disclosed in PCT Patent
Publication No. WO2004004632 and PCT Patent Publication No. WO2007148158 which are
incorporated herein by reference.
The general process for the preparation of the compound of formula I, or a pharmaceutically
acceptable salt thereof, comprises the following steps:
treating the resolved enantiomeric ally pure (-)-trans enantiomer
intermediate compound of formula VIA,
VIA
with acetic anhydride in the presence of a Lewis acid catalyst to obtain a resolved
acetylated compound of formula VIIA,
VIIA
b) reacting the resolved acetylated compound of formula VIIA with an acid of
formula ArCOOH or an acid chloride of formula ArCOCl or an acid anhydride of
formula (ArCO)20 or an ester of formula ArCOOCH3, wherein Ar is as defined
hereinabove in reference to the compound of formula I, in the presence of a base and a
solvent to obtain a resolved compound of formula VIIIA;
VIIIA
c) treating the resolved compound of formula VIIIA with a base in a suitable solvent to
obtain the corresponding resolved -diketone compound of formula IXA;
wherein Ar is as defined above;
d) treating the resolved -diketone compound of formula with an acid such as
hydrochloric acid to obtain the corresponding cyclized compound of formula XA,
e) subjecting the compound of formula XA to dealkylation by heating it with a dealkylating
agent at a temperature ranging from 120-180 °C to obtain the (+)-trans enantiomer of the
compound of formula I and, optionally, converting the subject compound into its
pharmaceutically acceptable salt.
The Lewis acid catalyst utilized in the step (a) above may be selected from: BF Et20 , zinc
chloride, aluminium chloride and titanium chloride.
The base utilized in the process step (b) may be selected from triethylamine, pyridine and a
DCC-DMAP combination (combination of N, N'-dicyclohexyl carbodiimide and 4-
dimethylaminopyridine) .
It will be apparent to those skilled in the art that the rearrangement of the compound of formula
VIIIA to the corresponding -diketone compound of formula is known as a Baker-
Venkataraman rearrangement (J. Chem. Soc, 1381 (1933) and Curr. Sci., 4,214 (1933)).
The base used in the process step (c) may be selected from: lithium hexamethyl disilazide,
sodium hexamethyldisilazide, potassium hexamethyldisilazide, sodium hydride and potassium
hydride. A preferred base is lithium hexamethyl disilazide.
The dealkylating agent used in process step (e) for the dealkylation of the compound of
formula may be selected from: pyridine hydrochloride, boron tribromide,boron
trifluoride etherate and aluminium trichloride. A preferred dealkylating agent is pyridine
hydrochloride.
Preparation of the starting compound of formula VIA involves reacting l-methyl-4-
piperidone with a solution of 1,3,5-trimethoxybenzene in glacial acetic acid, to yield
l-methyl-4-(2,4,6-trimethoxyphenyl)-l,2,3,6-tetrahydropyridine, which is reacted with boron
trifluoride diethyl etherate, sodium borohydride and tetrahydrofuran to yield 1- methyl-4- (2,4,6-
trimethoxyphenyl)piperidin-3-ol. Conversion of 1-methyl-4- (2,4,6- trimethoxyphenyl)piperidin-
3-ol to the compound of formula VIA involves converting the hydroxyl group present on the
piperidine ring of the compound, l-methyl-4-(2,4,6- trimethoxyphenyl)piperidin-3-ol to a
leaving group such as tosyl, mesyl, triflate or halide by treatment with an appropriate reagent
such as p-toluenesulfonylchloride, methanesulfonylchloride, triflic anhydride or
phosphorous pentachloride in the presence of oxygen nucleophiles such as triethylamine,
pyridine, potassium carbonate or sodium carbonate followed by ring contraction in the presence
of oxygen nucleophiles such as sodium acetate or potassium acetate in an alcoholic solvent
such as isopropanol, ethanol or propanol.
B) Preparation of (+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2- h drox methyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (compound A)
Molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added to (+)-trans-2-(2-chloro- phenyl)-
8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one (0.4 g, 0.9 mmol)
and heated at 180 °C for 1.5 h. The reaction mixture was cooled to 25 °C, diluted with MeOH
(10 mL) and basified using Na2C0 3 to pH 10. The mixture was filtered and the organic layer
was concentrated. The residue was suspended in water (5 mL), stirred for 30 min., filtered and
dried to obtain the compound, (+)-trans- 2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl- 1-methyl -pyrrolidin-3-yl)- chromen-4-one .
Yield: 0.25 g (70 ); IR (KBr): 3422, 3135, 1664, 1623, 1559 cm-1;
1H NMR (CDC13, 300MHz): 7.56 (d, 1H), 7.36 (m, 3H), 6.36 (s, 1H), 6.20 (s, 1H), 4.02 (m,
1H), 3.70 (m, 2H), 3.15 (m, 2H), 2.88 (m, 1H), 2.58 (s, 3H), 2.35 (m, 1H), 1.88 (m, 1H); MS
(ES+): m/z 402 (M+l);
Analysis: C2iH20ClNO5 C, 62.24 (62.71); H, 5.07 (4.97); N, 3.60 (3.48); CI, 9.01 (8.83).
The compound as obtained above (0.2 g, 0.48 mmol) was suspended in IPA (5 mL) and 3.5
% HC1 (25 ml) was added. The suspension was heated to get a clear solution. The solution was
cooled and solid filtered to obtain the compound, (+)-trans-2-(2- Chlorophenyl)-5,7-dihydroxy-
8-(2-hydroxymethyl- 1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride .
Yield: 0.21 g (97 ); mp: 188 - 192 °C ; [a]D25 = +21.3° (c = 0. 2, methanol);
1H NMR (CD30D, 300MHz): 7.80 (d, 1H), 7.60 (m, 3H), 6.53 (s, 1H), 6.37 (s, 1H), 4.23 (m,
1H), 3.89 (m, 2H), 3.63 (m, 1H), 3.59 (dd, 1H), 3.38 (m, 1H), 2.90 (s, 3H), 2.45 (m, 1H), 2.35
(m, 1H); MS (ES+): m/z 402 (M +1)( free base).
This compound was subjected to chiral HPLC. Chiral HPLC was done using column Chiralcel
OD-H (250 x 4.6 mm) and solvent system haxane:ethanol (92:08) with TFA (0.4%). The results
are recorded at 264nm with solvent flow rate of lmL/min. As depicted in the chiral HPLC
showed 100% e.e of the compound, (+)-trans-2-(2-chloro- phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-
1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
C) Preparation of (+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-
8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(Compound B)
A mixture of the compound, (+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-8-(2-
hydroxymethyl-1 -methyl pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one (0.25 g, 0.5 mmol),
pyridine hydrochloride (0.25 g, 2.16 mmol) and a catalytic amount of quinoline was
heated at 180 °C for a period of 2.5 hrs. The reaction mixture was diluted with methanol
(25 ml) and basified with solid Na2C0 3 to pH 10. The reaction mixture was filtered, and washed
with methanol. The organic layer was concentrated and the residue purified by column
chromatography using 0.1 % ammonia and 4.5 % methanol in chloroform as eluent to yield the
compound, (+)-trans-2-(2-chloro-4- trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-
l-methylpyrrolidin-3-yl)- chromen-4-one, as a yellow solid.
Yield: 0.15 g (63.7 %);
1H NMR (CDC13, 300MHz): 7.99 (m, 2H), 7.83 (d, 1H), 6.65 (s, 1H), 6.41 (s, 1H), 4.24 (m,
1H), 3.90 (m, 2H), 3.70 (m, 1H), 3.60 (m, 1H), 3.41 (m, 1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.28
(m, 1H); MS (ES+): m/z 470 (M+l).
The compound (0.1 g, 0.2 mmol) as obtained above was suspended in methanol (2 mL) and
treated with ethereal HC1 and the organic solvent evaporated to yield the compound, (+)-trans-2-
(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-
yl)-chromen-4-one hydrochloride.
Yield: 0.1g (92.8 %);
1H NMR (CDC13, 300MHz): 8.02 (d, 2H), 7.83 (d, 1H), 6.64 (s, 1H), 6.41 (s, 1H), 4.23 (m,
1H), 3.73 (m, 2H), 3.68 (m, 1H), 3.51 (m, 1H), 3.39 (m, 1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.31
(m, 1H).
IN VITRO STUDIES INVOLVING USE OF THE COMBINATION CONSISTING OF A
TDK INHIBITOR AND ONE OR MORE ANTINEOPLASTIC AGENTS
In vitro studies involving use of a combination comprising a CDK inhibitor selected from(+)-
trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl- pyrrolidin-3-yl)-
chromen-4-one hydrochloride (compound A) and (+)-trans-2-(2- chloro-4-trifluoromethylphenyl)-
5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl- pyrrolidin-3-yl)-chromen-4-one
hydrochloride (compound B) and one or more anti-neoplastic agents selected from
sorafenib, lapatinib, erlotinib, docetaxel, cisplatin or 5- fluorouracil, exhibiting the
synergistic effect of the combination of the present invention are illustrated in the
following examples.
Example 2:
Materials:
Sorafenib, lapatinib and erlotinib were obtained from LC Labs (USA). Cisplatin, 5- fluorouracil
and docetaxel were obtained from Sigma. CK-8 cytotoxicity kit was procured from Dojindo
Molecular Technologies, Japan. Culture media and fetal bovine serum (FBS) were obtained
from Sigma (St. Louis, MO) and Gibco (Paisley, Scotland) respectively. The head and neck
cancer cells SCC-25, Detroit 562, and FADU were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). Cells were maintained in Dulbecco's Modified Eagle
Medium (DMEM), supplemented with 10% FBS, Penicillin-Streptomycin Solution Stabilized,
sterile- filtered, with 100 units penicilin/ml and 100 mg streptomycin/ml. The cells were
grown in 75-cm culture flasks and kept in a humidified (37°C, 5% C0 2) incubator.
Cells were passaged on reaching 80% confluence.
Cell proliferation Assay:
Logarithmically growing cells were plated at a density of 3 x 10 cells/well and allowed
to recover overnight. The cells were challenged with varying concentration of different
anticancer agents (compound A, compound B, sorafenib, lapatinib erlotinib, cisplatin, docetaxel
and 5-fluorouracil) and the control cells received standard media containing dimethyl sulfoxide
(DMSO) vehicle at a concentration of 0.2%. After 72 hours, cell toxicity was determined by
CCK-8 (Cell Counting Kit-8 ) reagent (Dojindo Molecular Technologies, Japan); WST-8 (2-(2-
methoxy-4-nitrophenyl)-3-(4- nitrophenyl)-5-(2, 4-disulfophenyl)]-2H-tetrazolium, monosodium
salt) assay. In accordance with the manufacturer's instructions, 51 11 CCK-8 reagent was
added and plates were incubated for 2 hours. The toxicity was determined by measuring the
absorbance on Tecan Sapphire multi-fluorescence micro-plate reader (Tecan, Germany,
GmbH) at a wavelength of 450 nm corrected to 650 nm and normalized to controls.
A CCK-8 non-radioactive colorimetric assay was carried out to characterize the in vitro
growth of SCC-25, Detroit 562, and FADU as well as to test the anti
proliferative/cytotoxic activity of the anticancer agents, compound A, compound B ,
sorafenib, lapatinib, erlotinib, cisplatin, docetaxel and 5-fluorouracil when used in
combination. CCK-8 allows convenient assays using Dojindo's tetrazolium salt, WST [8[(2-(2-
methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- tetrazolium, monosodium
salt], which produces a water-soluble formazan dye upon bioreduction in the presence of an
electron carrier, 1-Methoxy PMS. CCK-8 solution is added directly to the cells; no pre-mixing
of components is required. CCK-8 is a sensitive nonradioactive colorimetric assay for
determining the number of viable cells in cell proliferation and cytotoxicity assays. WST-8
is bio-reduced by cellular dehydrogenases to an orange formazan product that is soluble
in tissue culture medium. The amount of formazan produced is directly proportional to the
number of living cells. The detection sensitivity of cell proliferation assays using WST-8 is
higher than assays using the other tetrazolium salts such as MTT, XTT, MTS or WST-1. Optical
Density was determined at measurement wavelength of 450 nm and reference wave length of
630 nm.
Determination of 50 percent inhibitory concentrations (IC 50) of the compound A,
compound B, sorafenib, lapatinib, erlotinib, docetaxel, cisplatin and 5-FU.
In order to determine the IC50 of compound A, compound B , sorafenib and lapatinib, in SCC-
25, Detroit 562 and FADU cells and IC50 of erlotinib, docetaxel, cisplatin and 5-FU in Detroit
562 and FADU cells, the cells were treated with the specified anticancer agents ("the test
compounds") at the below mentioned concentrations. All the anticancer agents in the following
doses of final concentration 0.03 , 0.1 ,0.3 , 1 , 3 , 10 , 30 and 100 
were analyzed for their capacity to exhibit cytotoxicity particularly to exhibit 50% cytotoxicity.
The cells were seeded at a density of 3000 cells/well, in a 200 ΐ in tissue culture grade 96
well plate and were allowed to recover for 24 hrs in a humidified 5% ± 0.2 C0 2 incubator at
37 °C ± 0.5 °C. After 24 hrs, 1 L• of 200 X (200 times higher than required concentration is
denoted as 200 X) test compound (compound A, compound B lapatinib, sorafenib, erlotinib,
docetaxel, cisplatin and 5-fluorouracil) dissolved in neat dimethyl sulfoxide (DMSO) was
added to the cells. The final DMSO concentration was 0.5% in wells. Plates were
incubated for 48 hrs in humidified 5% ± 0.2 C0 2 incubator at 37 ± 0.5 °C. After 48 hrs the
plates were removed from C0 2 incubator and 5 ΐ of Cell counting Kit (CCK-8) per well was
added. The same plate was kept at 37 °C for 3 hrs, and allowed to come to room temperature.
The absorbance at a wavelength of 450 nm was read on Tecan safire reader. The percent
cytotoxicity was calculated using the following formula.
Percent Cytotoxicity = (OP of Control - OP Treated cells X 100)
OP PMSO control
Pose response studies at 72 hr in SCC-25 cells showed that the Compound A, Compound B,
sorafenib and lapatinib inhibited 50% growth (IC50) at 0.4 , 1.1 , 2.7and 0.5
respectively. The results are presented in Table 1 and are graphically presented in Fig.
l a and lb.
Pose response studies at 72 hr in Petroit-562 cells showed that the Compound A, Compound B,
sorafenib and lapatinib inhibited 50% growth (IC50) at 1.3 , 14.1 , 6.1 and 3.9
respectively. The results are presented in Table 2 and are graphically presented in Fig. 2a
and 2b.
Pose response studies at 72 hr in FAPU cells showed that compound A, compound B, sorafenib
and lapatinib inhibited 50% growth (IC50) at 1.3 , 4.1 , 8.4 and 2.6
respectively. The results are presented in Table 3 and are graphically presented in Fig. 3a and
3b.
Pose-response studies at 72 hr in Petroit-562 cells showed that compound A and erlotinib
inhibited 50% growth (IC 50) at 1.3 , and 2.3 respectively. The results are presented in
Table 4 and are graphically presented in Fig. 4a.
Pose-response studies at 72 hr in FAPU cells showed that the compound A and erlotinib
inhibited 50% growth (IC 50) at 1.3 , and 10.2 respectively. The results are presented in
Table 5 and are graphically presented in Fig. 4b.
Pose-response studies at 72 hr in Petroit-562 cells showed that cisplatin, compound A and 5-FU
inhibited 50% growth (IC 50) at 14.6 , 1.3 , and 6.3 respectively. The results are
presented in Table 6 and are graphically represented in Fig. 5a.
Pose-response studies at 72 hr in FAPU cells showed that the cisplatin, compound A and 5-FU
inhibited 50% growth (IC 50) at 8.3 , 1.3 , and 11.6 respectively. The results are
presented in Table 7 and are graphically represented in Fig. 5b.
Dose-response studies at 72 hr in Detroit-562 cells showed that docetaxel, cisplatin,
compound A and 5-FU inhibited 10% growth (IC10) at 0.009 , 0.3, 0.1 and 0.3 
and 50% growth (IC50) at 0.85, 14.6, 1.3 , and 6.3 respectively. The results
are presented in Table 6 and are graphically represented in Fig. 5a.
Dose-response studies at 72 hr in FADU cells showed that docetaxel, cisplatin, compound A and
5-FU inhibited 10% growth (IC10) at 0.003, 0.25, 0.08 , and 0.31 and 50%
growth (IC 50) at 0.16 , 8.3 , 1.3 , and 11.6 respectively.
The results are presented in Table 7 and are graphically represented in Fig. 5b.
Similarly the IC30, IC70 and IC90 concentrations for all the tested compounds (anticancer
compounds) were established from dose in which particular compound shows 30 %, 70% and
90% activity respectively in the cytotoxicity assay.
Table 1 - 30 , 50%, 70% and 90% inhibitory concentrations (IC30, IC50, IC70
and IC90) of compound A, compound B, sorafenib and lapatinib in SCC-25 cells.
Table 2 - 30 %, 50%, 70% and 90% inhibitory concentrations (IC30, IC50, IC70
and IC90) of compound A, compound B, sorafenib and lapatinib in Detroit-562 cells.
Table 3 - 30 %, 50%, 70% and 90% inhibitory concentrations (IC30, IC50, IC70
and IC90) of compound A, compound B, sorafenib and lapatinib in FADU cells.
Table 4 - 30 %, 50%, 70% and 90% inhibitory concentrations (IC30, IC
and IC90) of compound A and erlotinib in Detroit-562 cells.
Table 5 - 30 %, 50%, 70% and 90% inhibitory concentrations (IC30, IC50, IC70
and IC90) of compound A and erlotinib in FADU cells.
Table 6 - 10%, 30 %, 50%, 70% and 90% inhibitory concentrations (ICio, IC30, IC50,
IC7oand IC90) of cisplatin, compound A, 5-FU and docetaxel in Detroit-562 cells.
Table 7 - 10%, 30 %, 50%, 70% and 90% inhibitory concentrations (ICio,IC 30, IC50,
IC oand IC90) of cisplatin, compound A , 5-FU and docetaxel in FADU cells
Example 4
Combination studies of compound A and sorafenib in SCC-25, Detroit-562 and
FADU cells.
A) SCC-25 cells
Sorafenib in the following dose of final concentration 0.18 and compound A in the following
doses of final concentration 0.1 , 0.4 and 4.1 were analyzed in single dose and in all
possible combinations of the dose range for the two anticancer agents mentioned above. The
sequence of treatment is as follows; the SCC-25 cells were treated with sorafenib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with plain MEM (minimum essential
media) medium. Fresh MEM with 10% serum (20011) was added, followed by treatment
with compound A from 24 hrs to 96 hrs. The results are presented in the following Table 8 and
graphically presented in figure 6a.
B) Detroit-562 cells.
Sorafenib in the following dose of final concentration 1.8 and compound A in the following
doses of final concentration 0.5 , 1.3 and 12.1 were analyzed in single dose and in all
possible combinations of the dose range for the two anticancer agents mentioned above. The
sequence of treatment is as follows; Detroit-562 cells were treated with sorafenib for 0 to 24 hrs.
At the end of 24 hrs the cells were washed two times with plain MEM medium. Fresh MEM
with 10% serum (20011) was added, followed by treatment with compound A from 24
hrs to 96 hrs. The results are presented in the following Table 9 and graphically presented in
figure 7a.
C) FADU cells
Sorafenib in the following dose of final concentration 3.9 and compound A in the following
doses of final concentration 0.2 , 1.3 and 8.3 were analyzed in single dose and in all
possible combinations of the dose range for the two anticancer agents mentioned above. The
sequence of treatment is as follows; the FADU cells were treated with sorafenib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with plain MEM medium. Fresh
MEM with 10% serum (200 ) was added, followed by treatment with compound A from
24 hrs to 96 hrs. The results are presented in the following Table 10 and graphically presented in
figure 8a.
Example 5
Combination studies of compound B and sorafenib in SCC-25, Detroit-562 and FADU
cells.
A) SCC-25 cells
Sorafenib in the following dose of final concentration 0.18 and compound B in the following
doses of final concentration 0.2 , 1.1 and 4.8 were analyzed in single dose and in all
possible combinations of the dose range for the two anticancer agents mentioned above. The
sequence of treatment is as follows; the SCC-25 cells were treated with sorafenib for 0 to 24 hrs.
At the end of 24 hrs the cells were washed two times with plain MEM medium. Fresh MEM
with 10% serum (200 ) was added, followed by treatment with compound B from 24 hrs
to 96 hrs. The results are presented in the following Table 1 1 and graphically presented in figure
6b.
B) Detroit-562 cells
Sorafenib in the following dose of final concentration 1.8 and compound B in the following
doses of final concentration 2.7 , 14.1 and 25.2 were analyzed in single dose and in
all possible combinations of the dose range for the two anticancer agents mentioned above. The
sequence of treatment is as follows; the Detroit-562 cells were treated with sorafenib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with plain MEM medium. Fresh
MEM with 10% serum (200 ) was added, followed by treatment with compound B from
24 hrs to 96 hrs. The results are presented in the following Table 12 and graphically presented in
figure 7b.
C) FADU cells
Sorafenib in the following dose of final concentration 3.9 and compound B in the following
doses of final concentration 2.3 , 4.1 and 9.6 were analyzed in single dose and in all
possible combinations of the dose range for the two drugs mentioned above. The sequence of
treatment is as follows; the FADU cells were treated with sorafenib for 0 to 24 hrs. At the
end of 24 hrs the cells were washed two times with plain MEM medium. Fresh MEM with
10% serum (200 ) was added, followed by treatment with compound B from 24 hrs to
96 hrs. The results are presented in the following Table 13 and graphically presented in figure
8b.
Example 6
Combination studies of compound A and lapatinib in SCC-25, Detroit-562 and FADU
cells.
A) SCC-25 cancer cells
Lapatinib in the following dose of final concentration 0.2 and compound A in the following
doses of final concentration 0.2, 0.5 and 3.3 were analyzed in single dose and in all
possible combinations of the dose range for the two drugs mentioned above. The sequence of
treatment is as follows; the SCC-25 cells were treated with lapatinib for 0 to 24 hrs. At the end
of 24 hrs the cells were washed two times with plain MEM medium. Fresh MEM with 10%
serum (200 ) was added, followed by treatment with compound A from 24 hrs to 96
hrs. The results are presented in the following Table 14 and graphically presented in figure 9a.
Sr. Anticancer agent (SCC-25 cells) %Cytotoxicity Combination
No. (Inhibitory cone, in ) index
1 Lapatinib IC30 17 -
2 Compound A IC30 12 -
3 Compound A IC50 23 -
4 Compound A IC70 26 -
5 Lapatinib IC30 + Compound A IC30 69 0.68
6 Lapatinib IC30 + Compound A IC50 83 0.71
7 Lapatinib IC30 + Compound A IC70 89 0.75
B) Detroit-562 cancer cells
Lapatinib in the following dose of final concentration 1 and compound A in the following
doses of final concentration 0.5 , 1.3 and 12.1 were analyzed in single dose and in all
possible combinations of the dose range for the two anticancer agents mentioned above. The
sequence of treatment is as follows; the Detroit-562 cells were treated with lapatinib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with plain MEM medium. Fresh
MEM with 10% serum (200 ) was added, followed by treatment with compound A from
24 hrs to 96 hrs. The results are presented in the following Table 15 and graphically presented in
figure 10a.
C) FADU cancer cells
Lapatinib in the following dose of final concentration 0.8 and compound A in the following
doses of final concentration 0.2 , 1.3 and 8.3 were analyzed in single dose and in all
possible combinations of the dose range for the two drugs mentioned above. The sequence of
treatment is as follows; the FADU cells were treated with lapatinib for 0 to 24 hrs. At the
end of 24 hrs the cells were washed two times with plain MEM medium. Fresh MEM with
10% serum (200 ) was added, followed by treatment with compound A from 24 hrs to
96 hrs. The results are presented in the following Table 16 and graphically presented in figure
11a.
Sr. Anticancer agent % Combination
No. (FADU cells) (Inhibitory cone, in ) Cytotoxicity index
1 Lapatinib IC30 19 -
2 Compound A IC30 13 -
3 Compound A IC50 22 -
4 Compound A IC70 24 -
5 Lapatinib IC30 + Compound A IC30 62 0.64
6 Lapatinib IC30 + Compound A IC50 85 0.69
7 Lapatinib IC30 + Compound A IC70 89 0.83
Example 7
Combination studies of compound B and lapatinib in SCC-25, Detroit-562 and FADU
cells.
A) SCC-25 cancer cells
Lapatinib in the following dose of final concentration 0.2 and compound B in the following
doses of final concentration 0.2 , 1.1 and 4.8 were analyzed in single dose and in all
possible combinations of the dose range for the two drugs mentioned above. The sequence of
treatment is as follows; the SCC-25 cells were treated with lapatinib for 0 to 24 hrs. At the end
of 24 hrs the cells were washed two times with plain MEM medium. Fresh MEM with 10%
serum (200 ) was added, followed by treatment with compound A from 24 hrs to 96
hrs. The results are presented in the following Table 17 and graphically presented in figure 9 b.
B) Detroit-562 cancer cells
Lapatinib in the following dose of final concentration 1.0 and compound B in the following
doses of final concentration 2.7 , 14.1 and 25.2 were analyzed in single dose and in
all possible combinations of the dose range for the two drugs mentioned above. The sequence of
treatment is as follows; the Detroit-562 cells were treated with lapatinib for 0 to 24 hrs. At the
end of 24 hrs the cells were washed two times with plain MEM medium. Fresh MEM with
10% serum (200 ) was added, followed by treatment with compound A from 24 hrs to
96 hrs. The results are presented in the following Table 18 and graphically presented in figure
10b.
C) FADU cancer cells
Lapatinib in the following dose of final concentration 0.8 and compound B in the following
doses of final concentration 2.3 , 4.1 and 9.6 were analyzed in single dose and in all
possible combinations of the dose range for the two anticancer agents mentioned above. The
sequence of treatment is as follows; the FADU cells were treated with lapatinib for 0 to 24
hrs. At the end of 24 hrs the cells were washed two times with plain MEM medium. Fresh
MEM with 10% serum (20011) was added, followed by treatment with compound A from
24 hrs to 96 hrs. The results are presented in the following Table 19 and graphically presented in
figure lib.
Example 8
Combination studies of compound A and erlotinib at IC30 concentration in Detroit-
562 cells
The combination of erlotinib and compound A was found to be synergistic at the IC30 of both
the anticancer agents. Erlotinib at IC30 showed cytotoxicity of 20.3% and Compound A at IC30,
showed cytotoxicity of 8.30%. However, when used as a combination of erlotinib IC30 for 24 hrs,
followed by compound A IC30 for 48 hrs showed an increase in cytotoxicity to the extent of 60%
was noted, which is 32% more cytotoxicity than the additive effect suggesting a synergistic
effect between the two anticancer agents in Detroit-562 cells with a combination index of 0.35.
The results are presented in the following Table 20 and graphically presented in figure 12a.
Example 9
Combination studies of compound A and erlotinib in FADU cells
The combination of erlotinib and Compound A was found to be synergistic at the IC30 of both the
anticancer agents erlotinib at IC30 showed cytotoxicity of 16% and Compound A at IC30,
showed cytotoxicity of 12.3%. However, when used as a combination of erlotinib at
concentration IC30 for 24hrs, followed by compound A at IC30 concentration for 48 hrs
showed an increase in cytotoxicity to the extent of 77% was noted, which is 49% more
cytotoxicity than the additive effect suggesting a synergistic effect between the two drugs in
FADU cells with a combination index of 0.23. The results are presented in the following
Table 2 1 and graphically presented in figure 12b.
In Vitro Studies Involving Use of Triple Combination Consisting of compound A. cisplatin
-
Example 10
Combination studies of compound A, cisplatin and 5-FU at the IC30 in Detroit-562 cells.
The combination of compound A and (cisplatin and 5-FU) was found to be synergistic at the
IC30 of each anticancer agents. Compound A at IC30 showed cytotoxicity of 10.4 % and
(cisplatin and 5-FU) at IC30, showed cytotoxicity of 28.60 %. However, when used as a
combination of (cisplatin and 5-FU) IC30 for 24hrs, followed by compound A IC30 for 48 hrs an
increase in cytotoxicity to the extent of 71% was noted, which was 33% more cytotoxicity than
the additive effect suggesting a synergistic effect between the three anticancer agents in
Detroit-562 cells with a combination index of 0.39. While the double combination Cisplatin
and 5-FU showed a combination index of 0.9. The results are presented in the following Table
22 and graphically presented in figure 13a.
Example 11
Combination studies of compound A, cisplatin and 5-FU at the IC30 in FADU cells.
The combination of compound A and (cisplatin and 5-FU) was found to be synergistic at the
IC30 of each anticancer agent. Compound A at IC30 showed cytotoxicity of 6.1% and (cisplatin
and 5-FU) at IC30, showed cytotoxicity of 30.1%. However, when used as a combination of
(cisplatin and 5-FU) at IC30 concentration for 24hrs, followed by compound A IC30 for 48hrs,
an increase in cytotoxicity to the extent of 81% was noted, which was 44% more cytotoxicity
than the additive effect suggesting a synergistic effect between the three drugs in FADU cells
with a combination index of 0.23. While the double combination cisplatin and 5-FU showed a
combination index of 0.89. The results are presented in the following Table 23 and graphically
presented in figure 13b.
Example 12
Combination studies of compound A, cisplatin and 5-FU with docetaxel at the IC30
concentration in Detroit-562 cells.
The combination of compound A and (cisplatin and 5-FU) with Docetaxel was found to be
synergistic at the IC3o of each anticancer agent. Compound A and Docetaxel at IC3o showed
cytotoxicity of 16.8% and 18.30 respectively (cisplatin and 5-FU) at IC3o, showed cytotoxicity
of 31.3%. However, when used as a combination of Docetaxel at IC3oconcentration for 12 hrs
followed by (cisplatin and 5-FU) at IC 0 concentration for 12hrs, followed by compound A at
IC3o concentration for 48hrs an increase in cytotoxicity to the extent of 96.38 % was noted,
with a combination index of 0.29. The results are presented in the following Table 24 and
graphically presented in figure 14a.
Example 13
Combination studies of compound A, cisplatin and 5-FU with docetaxel at the
IC30 concentration in FADU cells.
The combination of compound A and (cisplatin and 5-FU) with docetaxel was found to be
synergistic at the IC30 of each anticancer agent. Compound A and docetaxel at IC30
concentration showed cytotoxicity of 11.77% and 20.02 respectively (cisplatin and 5- FU) at
IC30 concentration, showed cytotoxicity of 51.39%. However, when used as a combination of
docetaxel at IC30 concentration for 12 hrs followed by (cisplatin and 5- FU) at IC30 concentration
for 12hrs, followed by compound A at IC30 concentration for 48 hrs an increase in
cytotoxicity to the extent of 98.24% was noted, with a combination index of 0.12. The
results are presented in the following Table 25 and graphically presented in figure 14b.
Example 14
Analysis of cleaved Caspase-3 expression levels
This study was conducted to evaluate the mechanisms by which the combination
consisting of sorafenib or lapatinib in combination with compound A or compound B blocks
proliferation and whether it can induce apoptosis in head and neck cancer cells. The cells were
seeded in 96-well plates at a density of 7.5 X 10 cells/well. 24 h post seeding, the minimum
essential medium was replaced with a fresh minimum essential medium with 10% serum. The
anticancer agents (sorafenib or lapatinib in combination with compound A or compound B)
were treated with specific concentration as mentioned below in SCC-25, Detroit-562 and
FADU cells and incubated for 48 hrs. At the end of 48 hrs, to determine the protein expression,
the cells were in 96 well plate spin down at 800g for 5 minutes. Culture supernatant was
removed and 200 ΐ of caspase-3 assay buffer was added and plates were again spin
down at 800g for 5 minutes. Supernatant were removed and cells were lysed with 100 ΐ
caspase-3 lysis buffer and incubated for 30 min in orbital shaker at 300 rpm at room
temperature. Further plates were spin down at 800g for 10 minutes and 90 ΐ of the
supernatant was transferred into new black well plate. To 90 ΐ of lysis solution 100 ΐ
of caspase-3 substrate was added and incubated for 30 minutes at 37'C. At the end of
incubation plates were read in Tecan Safire multimode reader with an excitation wavelength of
485 nm and emission wavelength of 535 nm.
A) Treatment pattern of sorafenib and compound A or compound B in SCC-25 cells for
assessing caspase-3 activity
The treatment with sorafenib for 24 hrs followed by either compound A or compound B for 48
hrs showed notable elevation of caspase3 expression than when used alone. It was also
observed that both compound A or compound B were more potent in inducing caspase-3
activity in combination as graphically represented in Fig 15a and Fig. 15b.
B) Treatment pattern of lapatinib and compound A or compound B in SCC-25 cells for
assessing caspase-3 activity
The treatment with lapatinib for 24 hrs followed by either compound A or compound B for 48
hrs showed notable elevation of caspase3 expression than when used alone. It was also
observed that both compound A or compound B were more potent in inducing caspase-3
activity in combination as graphically represented in Fig. 16a and Fig. 16b.
Example 15
In vivo efficacy studies in human head and neck cancer FaDu (Hypopharyngeal squamous
cell carcinoma) xenografts
In-vivo studies were carried out according to the method described in Clinical cancer search,
2003,9, 6052-6061; the disclosure of which is incorporated herein by reference for the teaching
of the assay.
Objective
The objective of this study was to evaluate the antitumor activity of Compound A in
combination with cetuximab or in combination with both, cisplatin and cetuximab in human
head and neck cancer xenograft model of FaDu.
The in-vivo studies were carried out using Xenograft models in Severe combined immune
deficiency (SCID) mice strain -CbySmn.CB17-Prkdcscid /J, by the method described below.
The statistically significant number of mice per group (n=6) was chosen in order to be able to
statistically evaluate the study data.
Method
FaDu cells were grown in MEM (minimum essential media) medium containing non-essential
amino acids and 10 % fetal calf serum in 5 % C02 incubator at 37 °C. Cells were pelleted by
centrifugation at 1000 rpm for 10 minutes. Cells were resuspended in pre-chilled mixture of
saline to get a count of 6 x 106 cells per mL; 0.2 ml of this cell suspension was kept on ice and
injected by subcutaneous (s.c.) route in SCID mice. Mice were observed every alternate day for
palpable tumor mass. Once the tumor size reached a size of 3-5 mm in diameter, animals were
randomized into respective groups of treatment and untreated controls. The treatment groups
comprised of 5 groups viz. 1) Compound A alone (Group 1); 2) cetuximab alone (Group 2); 3)
cisplatin alone (Group 3); 4) Compound A + cetuximab (Group 4); and 5) Compound A +
cisplatin + cetuximab (Group 5). The control group received no treatment. In single drug
treatment i.e. in respect of Groups 1, 2 and 3, the Compound A (35 mpk) was administered by
i.p route once daily for 5 days a week starting from day 1 of the week for 3 weeks with total of
15 doses; Cisplatin ( 1 mpk) was administered i.p. once a week on day 1 of the week with total of
3 doses. Cetuximab (2.5 mpk) was administered twice a week on days 1 and 4 of the week for 3
weeks with total of 6 doses. In the treatment with combination of drugs namely compound A
and cetuximab, the sequence that was followed included administration of Compound A for 2h
followed by cetuximab :
In the treatment with combination of drugs namely compound A, cisplatin and cetuximab, the
sequence that was followed included administration of cisplatin for 2h followed by the
Compound A for 2h, followed by cetuximab. Measurement of tumor was done every 2-3 days
apart. Growth inhibition percentage (GI ) was calculated at the end of experiment.
Terminal procedures:
At the end of the experiment, animals were euthanized using high dose of pentobarbital sodium
(100 mg/kg i.p./i.v.) or exposure to carbon dioxide gas.
Results
The results are as presented in Table 26 and graphically presented in Figure 17a. The Figure 17a
depicts the average group body weight over the period of drug (the therapeutic agents)
administration plotted. Figure 17b depicts the average % tumor weight of Head and Neck
carcinoma (Fadu) xenograft over a period of 18 days.
Table 26: Percent tumor growth inhibition at the end of treatment i.e. after 18 days.
The tumor growth inhibition was highly significant with p < 0.001 in the treatment groups
namely Group(s) 4 and 5 involving use of combination of antineoplastic agents with tumor
growth (TG) inhibition of 79% and. 77% respectively. There was no significant body weight loss
in all the treatment groups.
Conclusion
The combination of Compound A and cetuximab and the combination of Compound A,
cetuximab and cisplatin showed similar antitumor activity in the human head and neck cancer
xenograft model of FaDu and were significantly higher than either of the drugs alone.
Having thus described in detail various embodiments of the present invention, it is to be
understood that the invention defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent variations thereof are possible without
departing from the spirit or scope of the present invention.
We claim:
A pharmaceutical combination for use in the treatment of squamous cell carcinoma of
head and neck, wherein said pharmaceutical combination comprises a CDK inhibitor
selected from the compounds of formula I
wherein Ar is a phenyl group, which is unsubstituted or substituted by 1, 2, or 3
identical or different substituents selected from :halogen; nitro, cyano, Q-C-ralkyl,
trifluoromethyl, hydroxyl or C1-C4-alkoxy; or a pharmaceutically acceptable salt or
solvate thereof; and one or more antineoplastic agents selected from sorafenib, lapatinib,
erlotinib, cisplatin, 5-fluorouracil, docetaxel or cetuximab.
The pharmaceutical combination for the use according to claim 1, wherein in
the compound of formula I the phenyl group is substituted by 1, 2, or 3 identical
or different substituents selected from: chlorine, bromine, fluorine or iodine, Ci-C4-alkyl
or trifluoromethyl; or a pharmaceutically acceptable salt or solvate thereof.
The pharmaceutical combination for the use according to claim 2, wherein in
the compound of formula I the phenyl group is substituted by chlorine.
The pharmaceutical combination for the use according to claim 3, wherein the
compound of formula I is (+)-iraw5,-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-
1- methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound A).
The pharmaceutical combination for the use according to claim 2, wherein in
the compound of formula I the phenyl group is a substituted group substituted by
2 different substituents selected from chlorine and trifluoromethyl.
6. The pharmaceutical combination for the use according to claim 5, wherein the
compound of formula I is (+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-
dihydroxy- 8-(2-hydroxymethyl- 1-methyl-pyrrolidin- 3-yl)-chromen-4- one hydrochloride
(Compound B).
7. The pharmaceutical combination for the use according to any one of the preceding claims
1 to 6, wherein said antineoplastic agent is sorafenib.
8. The pharmaceutical combination for the use according to any one of the preceding claims
1 to 6, wherein said antineoplastic agent is lapatinib.
9. The pharmaceutical combination for the use according to any one of the preceding claims
1 to 6, wherein said antineoplastic agent is erlotinib.
10. The pharmaceutical combination for the use according to any one of the preceding claims
1 to 6, wherein said antineoplastic agents are cisplatin and 5-fluorouracil.
11. The pharmaceutical combination for the use according to any one of the preceding
claims 1 to 10, wherein said pharmaceutical combination further comprising radiation.
12. A pharmaceutical composition comprising a CDK inhibitor selected from the
compounds of formula I or a pharmaceutically acceptable salt or solvate thereof and
one or more antineoplastic agents selected from one or more of sorafenib,
lapatinib, erlotinib, cisplatin, 5-fluorouracil, docetaxel or cetuximab or pharmaceutically
acceptable salt thereof in association with a pharmaceutically acceptable carrier.
13. A method for the treatment of squamous cell carcinoma of head and neck in a
subject comprising administering to said subject a therapeutically effective amount of
a CDK inhibitor selected from the compounds of formula I ;
Formula I
wherein Ar is a phenyl group, which is unsubstituted or substituted by 1, 2, or 3
identical or different substituents selected from :halogen; nitro, cyano, Q-C-ralkyl,
trifluoromethyl, hydroxyl or Q-C-ralkoxy; or a pharmaceutically acceptable salt or
solvate thereof; in combination with a therapeutically effective amount of one or more
antineoplastic agents selected from sorafenib, lapatinib, erlotinib, cisplatin, 5-
fluorouracil , docetaxel or cetuximab.
14. The method according to claim 13, wherein the compound of formula I is (+)- ira ,-2-
(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-l-methyl-pyrrolidin-3-yl)-
chromen-4-one hydrochloride (Compound A).
15. The method according to claim 13, wherein the compound of formula I is (+)- trans-3-
[2[(2-Chloro-4-trifluoromethyl-phenyl) -5,7-dihydroxy- 8-(2-hydroxymethyl- 1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride (Compound B).
16. The method according to any one of the preceding claims 13 to 15, wherein said
antineoplastic agent is sorafenib.
17. The method according to any one of the preceding claims 13 to 15, wherein said
antineoplastic agent is lapatinib.
18. The method according to any one of the preceding claims 13 to 15, wherein said
antineoplastic agent is erlotinib.
19. The method according to any one of the preceding claims 13 to 15, wherein said
antineoplastic agents are cisplatin and 5- fluorouracil.
20. The method according to any one of the preceding claims 13 to 15, wherein said method
further comprising administration of radiation.